Kinases are a family of important signal-transducing enzymes that are activated when molecules bind to cell-surface receptors. Kinases phosphorylate cytoplasmic proteins, amplifying the binding event. There are currently no commercially available methods for imaging intracellular kinase activity. Imaging kinase activation in live cells could be useful for screening libraries of drug candidates for their ability to initiate signal transduction. To address this problem, the proposed Phase I research intends to investigate the use of reversible molecular sensors for imaging kinase activity in live cells. fluorescent, peptidic substrates for kinase will be synthesized and tested for response to phosphorylation in vitro and in vivo. The goal will be to design visible-wavelength, reversible kinase sensors useful for imaging the enzyme-mediated flux of phosphorylation. Initial enzyme targets will be protein kinase C (PKC), casein kinase Il (CklI) and tyrosine kinase. The result will be a method similar to that used for the imaging the flux of intracellular Ca2+ and will be applicable to imaging kinase activity in both single cells and populations of cells. Reversible kinase sensors could offer important insights into signal transduction pathways and also serve as powerful tools for high throughput screening and drug discovery. PROPOSED COMMERCIAL APPLICATIONS: Reagents proposed herein will result in assays for high-throughput screening, sensors for imaging kinase activity in live cells and methods for the detection of kinase activity in vitro.